Method for producing vitamin K2 from culture of Bacillus natto

ABSTRACT

A method for producing vitamin K2 comprising: culturing  Bacillus natto  in a liquid medium to obtain a  Bacillus natto  culture or culture supernatant thereof, wherein the culture or culture supernatant contains vitamin K2; treating the culture or culture supernatant with at least one coagulant selected from the group consisting of an inorganic coagulant and a cationic polymer coagulant, with the proviso that the coagulant excludes chitosan, so as to obtain a treated coagulant to which vitamin K2 is absorbed or aggregated; separating the treated coagulant from the treated culture or culture supernatant, and obtaining an oily product that contains vitamin K2 from the separated coagulant.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/296,942 filed Dec. 8, 2005, entitled “Method for ProducingFoods from Culture of Bacillus Natto”, which claimed priority toJapanese Patent Application No. 2004-378455 filed Dec. 28, 2004.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for producing a food productcontaining a thrombolytic enzyme, nattokinase, but containing little ornone of a blood coagulation factor, vitamin K2, in particular, a foodproduct made from a culture of Bacillus natto.

The present invention also relates to a method for producing vitamin K2from a culture of Bacillus natto.

2. Description of Related Art

It was discovered by Sumi et al. that the Bacillus natto produces thethrombolytic enzyme nattokinase (Experientia Vol. 43, pp. 1110-1111(1987)). The nutritional value of natto (fermented soybeans) and itsvalue as a health food have been highly evaluated. It is known thatnattokinase itself acts as a fibrinolytic enzyme. Nattokinase lysesthrombi when natto is ingested as a food, thus nattokinase has thethrombolytic activity. Nattokinase has extremely good characteristicssuch as having a long half life and retaining its effectiveness for along period of time. In addition, it has been reported that nattokinasehas a therapeutic effect on incipient central retinal vein occlusionalthough the effect cannot be clearly seen with urokinase, which alsohas the thrombolytic activity (Nishimura et al., Japanese Review ofClinical Ophthalmology Vol. 88, No. 9, pp. 53-57 (1994)).

Accordingly, food products containing large amounts of nattokinase, forexample, powders and encapsulated products made from Bacillus nattoculture have been marketed as health foods.

On the other hand, Bacillus natto is known to product large amounts ofvitamin K2. Vitamin K2 is known as an essential element in the bloodcoagulation system. Vitamin K2 has other physiological functions, and issaid to cause absorption difficulties in newborns and osteoporosis inthe elderly when it is deficient, and is said to cause disorders such ashemolytic anemia, splenomegaly, nephropathy, and hepatopathy when it ispresent in excess. Thus, a Bacillus natto culture extract contains notonly nattokinase as an effector of thrombolytic system but also vitaminK2 as an effector of blood coagulation system.

The required daily intake of vitamin K's for adults is generally 55 to65 μg. Large amounts of vitamin K's are contained in foods such asseaweed, broccoli and the like. Vitamin K's are produced byenterobacteria. Also, vitamin K's can be produced by Bacillus natto thatgrow in the intestines with ingesting natto. Therefore, it is said thatthe required intake of vitamin K2 can be satisfied with a normal diet.Thus, it is generally not necessary to supplement vitamin K2.

Further, problems regarding ingesting natto would occur with patientswho receive inhibitors for the vitamin K-dependent blood coagulationfactors such as prothrombins VII, IX, and X to prevent thrombosis. Ifthe patients ingest a natto or a Bacillus natto culture extract, each ofwhich contains the thrombolytic enzyme nattokinase, for example, inorder to prevent thrombosis, they also would intake vitamin K2 at thesame time, and the effects of the inhibitors for the vitamin K-dependentblood coagulation factors would be counteracted.

Accordingly, in order to prevent thrombogenesis, it is desirable toobtain a food product made from Bacillus natto culture extract in whichvitamin K2 has been reduced. Therefore, various methods have beenattempted for reducing vitamin K2 in the food product. These methodsinclude extraction of fat soluble vitamin K2 using organic solvents suchas hexane. However, there have been some problems in this extractionmethod as described below. Fat soluble nutrients as well as vitamin K2would be extracted out and reduced in the food product. The need toremove organic solvents would lead to an increase in the cost ofmanufacturing. The organic solvents would remain in the food products.The use of organic solvents would cause consumers to resist againstingestion.

A method for removing vitamin K2 using chitosan has been proposed (U.S.Pat. No. 6,730,504). However, chitosan must be dissolved in water beforetreatment, and is expensive. Therefore, there is a demand foralternative methods for removing vitamin K2.

SUMMARY OF THE INVENTION

The present invention has an object to provide a method for producing afood product from Bacillus natto culture from which vitamin K2 has beenremoved.

The present invention provides a method for producing a food productfrom Bacillus natto culture, said food product containing nattokinaseand about 1 μg or less of vitamin K2/g dry weight. The method comprisestreating a Bacillus natto culture or culture supernatant with at leastone coagulant selected from the group consisting of an inorganiccoagulant and a cationic polymer coagulant, with the proviso that thecoagulant excludes chitosan.

In an embodiment, the inorganic coagulant is at least one inorganiccoagulant selected from the group consisting of calcium chloride andpoly aluminum chloride.

In a further embodiment, the inorganic coagulant and a phosphoruscoagulant are added to the Bacillus natto culture or culturesupernatant.

In another embodiment, the cationic polymer coagulant is at least oneselected from the group consisting of a cationic polysaccharide, ahexamethylenediamine/epichlorohydrin polycondensate and adimethylamine/epichlorohydrin polycondensate.

In a further embodiment, the cationic polymer coagulant and apolyacrylate are added to the Bacillus natto culture or culturesupernatant.

In one embodiment, the food product is in the form of one selected fromthe group consisting of concentrate, paste, powder, granule, capsule,drinkable preparation and tablet.

The present invention further provides a method for producing vitamin K2comprising: culturing Bacillus natto in a liquid medium to obtain aBacillus natto culture or culture supernatant thereof, wherein theculture or culture supernatant contains vitamin K2; treating the cultureor culture supernatant with at least one coagulant selected from thegroup consisting of an inorganic coagulant and a cationic polymercoagulant, with the proviso that the coagulant excludes chitosan, so asto obtain a treated coagulant to which vitamin K2 is absorbed oraggregated; separating the treated coagulant from the treated culture orculture supernatant, and obtaining an oily product that contains vitaminK2 from the separated coagulant.

In an embodiment, the separation is performed by filtration so as toobtain a filtration residue, and vitamin K2 is extracted with an organicsolvent from the filtration residue.

The present invention further provides an oily product that containsvitamin K2 obtained by the above mentioned method for producing vitaminK2.

According to the method of the present invention, a food product fromBacillus natto culture which contains nattokinase and about 1 μg or lessof vitamin K2/g dry weight can be obtained in a simple manner. Since thefood product contains only extremely little of vitamin K2, it canprevent excessive intake of vitamin K2 and thrombogenesis in healthypeople. Further, the food product is excellent in that people withrestriction of vitamin K2 intake can also safely intake nattokinase.

DETAILED DESCRIPTION OF THE INVENTION

A method for producing a food product from Bacillus natto cultureaccording to the present invention includes a step of treating aBacillus natto culture (preferably liquid culture) or culturesupernatant with a specific agent to substantially remove vitamin K2 inthe culture or culture supernatant. Hereinafter, the method of thepresent invention will be described. Since numerical values of vitaminK2, etc. can vary with any factors such as the type and the culturecondition of Bacillus natto used, the present invention should not belimited by specific numerical values of vitamin K2, etc. describedherein.

Culturing of Bacillus Natto

Any microorganisms that are classified as Bacillus natto and that canproduce nattokinase can be used in the method of the present invention,including Bacillus natto which can be isolated from commerciallyavailable “natto”, fermented soybeans. For example, Bacillus subtilisnatto can be used. Hereinafter, any microorganisms which can be used inthe method of the present invention will be collectively referred to as“Bacillus natto”.

The culture medium used for Bacillus natto should not be limited. Theculture medium may be a liquid medium. It is preferable to select amedium by considering that the concentrated medium will be applied as afood product. In the culture medium for Bacillus natto, carbon sourcematerials such as starch (for example, corn starch), glucose, andsucros; nitrogen source materials such as defatted soybeans and meatextract; inorganic salts such as calcium carbonate and magnesiumchloride can be contained. Fatty acids and the like can be optionallycontained in the culture medium. It is preferable that the components ofthe culture medium are food additive grade.

The method for culturing Bacillus natto should not be limited, andincubation with aeration and agitation is preferable for large scaleculture. The incubation temperature is not limited as far as Bacillusnatto can grow. The temperature is preferably about 30 to 45° C., morepreferably about 32 to 42° C., and most preferably approximately 37° C.It is preferable that the incubation period be about 3 to 4 days.

Generally, the supernatant after incubation of Bacillus natto caninclude approximately 300 to 600 FU/ml of nattokinase, and approximately10 to 100 μg of vitamin K2/g dry weight.

Treatment for Removing Vitamin K2

Vitamin K2 can be removed from the culture or culture supernatant usingat least one coagulant selected from the group consisting of aninorganic coagulant and a cationic polymer coagulant (excludingchitosan).

Inorganic Coagulant

The inorganic coagulant includes, but is not limited to, calciumchloride, poly aluminum chloride, aluminum sulfate, ferric chloride,polysilicate, and ferrous sulfate. As the inorganic coagulant, calciumchloride, disodium hydrogenphosphate, and poly aluminum chloride can bepreferably used. In order to enhance the coagulation by the inorganiccoagulant, a combination of the inorganic coagulant with a phosphoruscoagulant (e.g., disodium hydrogenphosphate, sodium dihydrogenphosphate,potassium dihydrogenphosphate or tripotassium phosphate) can be used.For example, a combination of calcium chloride and disodiumhydrogenphosphate and a combination of poly aluminum chloride anddisodium hydrogenphosphate can be preferably used. When using acombination of coagulants, the coagulants may be mixed before theiradditions, or alternatively, one of the coagulants may be added first,and then the other coagulant may be added. For example, when using acombination of calcium chloride and disodium hydrogenphosphate as acoagulant, disodium hydrogenphosphate can be added first, and thencalcium chloride can be added.

Poly aluminum chloride is a compound that is represented by a generalformula [Al₂(OH)_(n)Cl_(6-n)]_(m) and compounds in the range of 1≦n≦5and m≦10 can be preferably used. Poly aluminum chloride is commerciallyavailable, and for example, it is available from TAKI CHEMICAL CO., LTD.

The inorganic coagulant can be added to a culture, which containsmicroorganisms, or to a culture supernatant, which is obtained byremoving microorganisms by means of separation such as centrifugation orfiltration. The inorganic coagulant can be added to the culture or theculture supernatant as it is, or in the form of liquid in which theinorganic coagulant is dissolved or dispersed in water. It is preferableto add the inorganic coagulant in the form of liquid. The amount of theinorganic coagulant added can be without limitation and determineddepending on the type of the coagulant. The pH in the coagulationreaction can be adjusted as appropriate before or after addition inaccordance with the inorganic coagulant used.

As an example, use of a combination of calcium chloride and disodiumhydrogenphosphate to treat a Bacillus natto culture or culturesupernatant will be described below. Disodium hydrogenphosphate ispreferably added in an amount of about 1.5 to 7.5 parts by weight, morepreferably about 4 to 5 parts by weight per 100 parts by weight of theculture or culture supernatant. Calcium chloride is preferably added inan amount of about 0.5 to 2.5 parts by weight, more preferably about 1to 1.8 parts by weight per 100 parts by weight of the culture or culturesupernatant. For example, disodium hydrogenphosphate is added anddissolved first, and then calcium chloride is added. Thereafter, the pHof the culture or culture supernatant is adjusted preferably in therange of about 7 to 7.4

When treating a Bacillus natto culture or culture supernatant with polyaluminum chloride, poly aluminum chloride is added preferably in anamount of about 0.001 to 0.6 parts by weight (about 10 to 6,000 ppm perthe solution), more preferably about 0.3 to 0.5 parts by weight (about3,000 to 5,000 ppm per the solution) per 100 parts by weight of theculture or culture supernatant. The pH of the culture or culturesupernatant to which poly aluminum chloride has been added is preferablyadjusted in the range of about 4.5 to 5.0.

Cationic Polymer Coagulant

The cationic polymer coagulant includes, but is not limited to, cationicpolysaccharides, hexamethylenediamine/epichlorohydrin polycondensates,dimethylamine/epichlorohydrin polycondensates, water-soluble anilineresin hydrochloride, polyethyleneimine, polyamine,polydianildimethylammonium chloride, and hexamethylenediamine. However,the cationic polymer coagulant does not include chitosan. In particular,cationic polysaccharides, hexamethylenediamine/epichlorohydrinpolycondensates, and dimethylamine/epichlorohydrin polycondensates canbe preferably used. A polyacrylate can be added to the cationic polymercoagulant to enhance the coagulation. For example, a combination ofcationic polysaccharides and polyacrylates is preferably used. Acationic polymer coagulant and a polyacrylate may be mixed beforeadditions thereof, or alternatively either one of them may be addedfirst and then the other may be added.

An example of cationic polysaccharides is Kiprogum NGK (trade name:manufactured by NIPPON STARCH CHEMICAL CO., LTD.).

The cationic polymer coagulant can be added to a culture or culturesupernatant as it is, or in the form of liquid in which the cationicpolymer coagulant is dissolved or dispersed in water. The amount of thecationic polymer coagulant added can be without limitation anddetermined depending on the type of the cationic polymer coagulant. ThepH in the coagulation reaction can be adjusted as appropriate before orafter the addition in accordance with the cationic polymer coagulantused.

As an example, use of cationic polysaccharides,hexamethylenediamine/epichlorohydrin polycondensates, ordimethylamine/epichlorohydrin polycondensates to treat a Bacillus nattoculture or culture supernatant will be described below.

Cationic polysaccharides are preferably added in an amount of about0.005 to 0.2 parts by weight, more preferably about 0.02 to 0.04 partsby weight per 100 parts by weight of the culture or culture supernatant.The pH of the culture or culture supernatant is preferably adjustedbefore addition of cationic polysaccharides. The pH is preferably in therange of about 4 to 5, more preferably about 4.5.

The cationic polysaccharides are preferably combined with sodiumpolyacrylate. They can be used for the treatment in a combination ofamounts within the ranges described herein.

The polyacrylate includes, but is not limited to, sodium polyacrylate,potassium polyacrylate, ammonium polyacrylate, lithium polyacrylate, andamine salts of polyacrylate.

Sodium polyacrylate is preferably added in an amount of about 0.005 to0.2 parts by weight, more preferably about 0.02 to 0.04 parts by weightper 100 parts by weight of the culture or culture supernatant. The pH ofthe culture or culture supernatant is preferably adjusted beforeaddition of sodium polyacrylate. The pH is preferably in the range ofabout 4 to 5, more preferably about 4.5.

A hexamethylenediamine/epichlorohydrin polycondensate is preferablyadded in an amount of about 0.0001 to 0.02 parts by weight (about 1 to200 ppm per the solution), more preferably about 0.005 to 0.01 parts byweight (about 50 to 100 ppm per the solution) per 100 parts by weight ofthe culture or culture supernatant. The pH of the culture or culturesupernatant to which hexamethylenediamine/epichlorohydrin polycondensatehas been added is adjusted preferably in the range of about 6.5 to 7.5,more preferably about 7.

A dimethylamine/epichlorohydrin polycondensate is preferably added in anamount of about 0.0001 to 0.02 parts by weight (about 1 to 200 ppm perthe solution), more preferably about 0.005 to 0.01 parts by weight(about 50 to 100 ppm per the solution) per 100 parts by weight of theculture or culture supernatant. The pH of the culture or culturesupernatant to which dimethylamine/epichlorohydrin polycondensate hasbeen added is preferably adjusted in the range of about 6.5 to 7.5, morepreferably about 7.

Removal of Coagulant

The coagulant is added to the Bacillus natto culture or culturesupernatant, and the mixture is stirred extensively and then filtrated,for example, using a pressure filtering apparatus, with a filter aidsuch as pearlite and diatomite to obtain a clear filtrate.Alternatively, the coagulant is added to the culture or culturesupernatant, and then a filter aid such as pearlite and diatomite isadded thereto, and then the mixture is stirred for an appropriate timeand then filtrated, for example, using a pressure filtering apparatus,to obtain a clear filtrate. The filtrate thus obtained may be furthertreated while, if necessary, adjusting the pH. By treating the Bacillusnatto culture or culture supernatant with the coagulant, at least about95%, preferably at least about 99%, and more preferably at least about99.9%, of the vitamin K2 can be removed, while nattokinase is not almostremoved and remains in the filtrate.

Although the coagulant is dissolved or dispersed in the culture orculture supernatant, it is removed by the filter aid, and is not almostcontaminated in the filtrate. It is believe that because the coagulantis adsorbed to microorganisms or some components in the culture orculture supernatant, it can be removed by the filter aid. The coagulantand/or filter aid to which vitamin K2 and Bacillus natto cells have beenabsorbed or aggregated can be collected as a filtration residue.

Food Product

If necessary, the filtrate is further subjected to subsequent treatment,such as micro filtration with a filter aid. Then the filtrate isconcentrated using a concentrator, such as a reverse osmosisconcentrator, to obtain a concentrate. Almost all the substances havinga molecular weight of 100 or less can be removed by the reverse osmosisconcentrator. Optionally, the concentrate may be subjected to sterilefiltration through a membrane filter, for example, having a pore size ofabout 0.5 μm or about 0.2 μm. The concentration of vitamin K2 in theconcentrate can be below 1 μg/g dry weight.

The concentrate is further concentrated into a paste. To the concentratea food additive such as water-soluble dietary fibers, lactose, celluloseor the like is added in an appropriate amount, and the mixture isfreeze-dried to form a powder or granule. The concentrate, paste, poweror granule can be encapsulated. The concentrate, paste, power or granulecan be used for producing a tablet. The tablet can be various types ofsurface-coated tablets, including a sugar-coated pill, depending ontheir use. Thus, the food product of the present invention can beproduced.

The food product contains nattokinase, but can contain vitamin K2 onlyin an amount of below about 1 μg/g dry weight. The amount of vitamin K2in the food product can be below the detection limit (about 0.001 μg/gdry weight). The content of nattokinase is without limitation, andpreferably about 20 FU or more, more preferably about 1000 FU or more,and even more preferably about 2500 FU or more, per 1 g. The content ofnattokinase can vary with the form of the food product. In the case ofdry powder, the content can be about 5000 FU or more per 1 g, and can beabout 10000 FU or more per 1 g. The activity of nattokinase can bedetected, for example, by detecting any plaque formed on a fibrin plateaccording to the method as described in Experientia Vol. 43, pp.11190-1111 (1987). In the present invention, nattokinase refers to anenzyme that is produced by Bacillus natto and has an ability of forminga clear plaque on a fibrin plate.

The activity of nattokinase and the amount of vitamin K2 can bedetermined in the following manner.

A: Measurement of Nattokinase Activity

Nattokinase is allowed to act upon fibrin, and the amount ofacid-soluble low molecular degradation product is increased withdegradation of fibrin. The increase in the degradation product isdetermined by measuring the absorbance of an ultraviolet ray (275 nm).

a-1: Preparation of an Aqueous Fibrinogen Solution

Seventy-two mg of fibrinogen (produced by Sigma Corp., fibrinogenfraction I derived from bovine blood plasma, Type I-S) is dissolved in10 ml of 50 mM borax buffer (pH 8.5; containing 150 mM NaCl) to preparea 0.72% (w/v) aqueous fibrinogen solution.

a-2: Preparation of a Thrombin solution

Thrombin (produced by Sigma Corp., derived from bovine blood plasma) isdissolved in 50 mM borax buffer so as to have a concentration of1000/ml. When this solution is to be used, it is diluted 50 times (i.e.,into 20 U/ml) with the borax buffer.

a-3: Activity Measurement

First, 1.4 ml of 50 mM borax buffer and 0.4 ml of aqueous fibrinogensolution are placed into a test tube, and then warmed at 37° C.±0.3° C.in a water bath for five minutes. Then, 0.1 ml of the thrombin solutionis added thereto, and the mixture is stirred. This mixture is allowed tostand in the water bath for 10 minutes. Then, 0.1 ml of a samplesolution is added to the mixture, and the mixture is stirred for 5seconds, and then allowed to stand in the water bath. The mixture isstirred for 5 seconds at 20 minutes and 40 minutes after adding thesample solution. Sixty minutes after adding the sample solution, 2 ml of0.2 M trichloroacetate solution is added thereto, and the mixture isstirred, and allowed to stand for an additional 20 minute period. Thereaction mixture is centrifuged for 5 minutes at 15,000×g, and theabsorbance (Ar) of the supernatant at 275 nm is measured.

As a control, 1.4 ml of 50 mM borax buffer and 0.4 ml of aqueousfibrinogen solution are placed into a test tube, and then warmed at 37°C.±0.3° C. in a water bath for five minutes. Then, 0.1 ml of thethrombin solution is added, and the mixture is stirred. This mixture isallowed to stand in the water bath for 10 minutes. Then, 2 ml of 0.2 Mtrichloroacetate solution is added thereto, and the mixture is stirred.Next, 0.1 ml of the sample solution is added to the mixture and stirred,and allowed to stand for 20 minutes. The reaction mixture is centrifugedfor 5 minutes at 15,000×g, and the absorbance (Ac) of the supernatant at275 nm is measured.

Nattokinase activity is determined according to the formula below:A(FU/ml)={(Ar−Ac)/(0.01×60×0.1)}×D,

-   -   where D is the dilution ratio.        B. Quantification of Vitamin K2

As described below, a sample is prepared for HPLC measurement andvitamin K2 is measured by HPLC.

b-1: Preparation of a Sample for HPLC Measurement

First, 0.5 ml of a sample to be measured, 0.5 ml of water, and 1.5 ml ofisopropanol are mixed and stirred, and then 5 ml of hexane is addedthereto, and is stirred. The mixture is centrifuged at 1700×g for 10minutes at 20° C. to give 4 ml of the supernatant (organic layer). Thesupernatant is concentrated, dried, and then dissolved in 100 μl ofethanol to give the solution of the sample.

B-2: HPLC Conditions

The HPLC conditions are as follows:

-   -   Column: Shimadzu STR ODS-2 4.6×250 mm    -   Eluant: 97% ethanol    -   Flow rate: 0.7 ml/min.    -   Detection: UV 254 nm

Under these conditions, the retention time of vitamin K2 is between 16and 17 minutes.

Production of Vitamin K2

According to the invention, Bacillus natto is cultured in a liquidmedium to allow it to produce vitamin K2. Therefore, a Bacillus nattoculture or culture supernatant thereof contains vitamin K2. Thecultivation of Bacillus natto is as described above. The culture orculture supernatant is treated with at least one coagulant selected fromthe group consisting of an inorganic coagulant and a cationic polymercoagulant, with the proviso that the coagulant excludes chitosan, toseparate vitamin K2. Since vitamin K2 can be absorbed or aggregated tothe coagulant, vitamin K2 can be separated and collected by thesubsequent recovery of the coagulant from the treated culture or culturesupernatant. A filter aid can also be used to help the subsequentrecovery of the coagulant. Therefore, preferably, the treated culture orculture supernatant is filtered to obtain a filtration residue thatincludes the coagulant and vitamin K2. From the filtration residue,vitamin K2 is extracted with an organic solvent. Alternatively, thetreated culture or culture supernatant is centrifuged to obtain aprecipitate including the coagulant and vitamin K2, and from theprecipitate vitamin K2 is extracted with an organic solvent.

The extraction can be performed by adding an organic solvent to thefiltration residue or the precipitate. Examples of the organic solventinclude lower alcohols (such as methyl alcohol, ethyl alcohol, butylalcohol, propyl alcohol and isopropyl alcohol), hexane, ethyl ether,acetone, chloroform, ethyl acetate and any combinations thereof. As theorganic solvent, hexane-isopropyl alcohol (at the ratio of 3:2 byweight) is preferably used. Then, the extract is collected and thesolvent is removed so as to obtain an oily product that contains vitaminK2. Further, vitamin K2 can be purified from this oily product, usingany methods commonly used by those skilled in the art such as moleculardistillation and steam distillation. Both the oily product that containsvitamin K2 and purified vitamin K2 can be formulated into the form suchas oil, powder, granule, or tablet. The oily product that containsvitamin K2 or purified vitamin K2 can be applied in the food orpharmaceutical industry, and for medical treatments such as preventionof osteoporosis.

EXAMPLES

The following examples are provided to explain the present invention. Itshould be appreciated that the present invention is not limited thereby.

Example 1

A culture medium at pH 7 containing 1 wt % of polypeptone, 1 wt % ofglucose, 0.5 wt % meat extract, and 0.2 wt % of NaCl was placed in around bottom flask, inoculated with Bacillus natto isolated from nattothereto, and then incubated at 37° C. for 18 hours. The Bacillus nattoculture thus obtained was inoculated into a seed culture tank containingthe culture medium having the same composition as the culture medium inthe round bottom flask, and incubated for 22 hours to give a Bacillusnatto seed culture.

Then, a culture medium at pH 7.3 containing 6.25 wt % of corn starch,3.09 wt % of defatted soybeans, 0.15 wt % of food additive grade calciumcarbonate, 1.5 wt % of soybean oil, and 0.008 wt % of silicone wasprepared. To the culture medium at pH 7.3, the Bacillus natto seedculture was added, and then incubated at 37° C. for 70 hours withaeration of 0.5 VVM. The resultant Bacillus natto culture contained 480FU/ml of nattokinase and 5.7 μg of vitamin K2 per gram of culture.

Then, 4.4 g of disodium hydrogenphosphate was added to 100 g of theresultant Bacillus natto culture, and the mixture was stirredextensively, and then 3.3 g of a 35 wt % calcium chloride aqueoussolution and 2.7 g of pearlite were added thereto and the mixture wasstirred at room temperature for 30 minutes. The pH of the culture wasadjusted to 7.2, and the culture was filtrated under reduced pressure togive a filtrate. The activity of nattokinase and the amount of vitaminK2 in the filtrate were determined according to the procedures A and Bdescribed above, respectively. The results are shown in Table 1.

Example 2

First, 4.5 g of a 10 wt % of poly aluminum chloride aqueous solution and1.8 g of pearlite were added to 100 g of the resultant Bacillus nattoculture in Example 1, and the mixture was stirred at room temperaturefor 10 minutes. The pH of the culture was adjusted to 5.0, and theculture was filtrated under reduced pressure to give a filtrate. Theactivity of nattokinase and the amount of vitamin K2 in the filtratewere determined as in Example 1. The results are shown in Table 1. Thepoly aluminum chloride was obtained from TAKI CHEMICAL CO., LTD.

Example 3

The pH of 100 g of the resultant Bacillus natto culture in Example 1 wasadjusted to 4.5, and 0.04 g of sodium polyacrylate (trade name: “AQUALICFH” manufactured by NIPPON SHOKUBAI), 0.02 g of cationic polysaccharides(trade name: “Kiprogum NGK” manufactured by NIPPON STARCH CHEMICAL CO.,LTD.) and 2.5 g of pearlite were added thereto, and the mixture wasstirred at room temperature for 10 minutes and subjected to filtrationunder reduced pressure to give a filtrate. The activity of nattokinaseand the amount of vitamin K2 in the filtrate were determined as inExample 1. The results are shown in Table 1.

Example 4

First, 10 g of an aqueous solution containing 0.2 wt % of a 30%hexamethylenediamine/epichlorohydrin polycondensate aqueous solution(trade name: “SANPOLY K-601” manufactured by SANKYO CHEMICAL INDUSTRIES,LTD.), and 2.5 g of pearlite were added to 100 g of the resultantBacillus natto culture in Example 1, and the mixture was stirred at roomtemperature for 10 minutes. The pH of the culture was adjusted to 7.0,and the culture was filtrated under reduced pressure to give a filtrate.The activity of nattokinase and the amount of vitamin K2 in the filtratewere determined as in Example 1. The results are shown in Table 1.

Example 5

First, 10 g of an aqueous solution containing 0.2 wt % of a 50%dimethylamine/epichlorohydrin polycondensate aqueous solution (tradename: “SANPOLY K-108” manufactured by SANKYO CHEMICAL INDUSTRIES, LTD.),and 2.5 g of pearlite were added to 100 g of the resultant Bacillusnatto culture in Example 1, and the mixture was stirred at roomtemperature for 10 minutes. The pH of the culture was adjusted to 7.0,and the culture was filtrated under reduced pressure to give a filtrate.The activity of nattokinase and the amount of vitamin K2 in the filtratewere determined as in Example 1. The results are shown in Table 1.

Comparative Example 1

Chitosan (manufactu8red by KYOWA TECNOS CO., LTD.) was dissolved in 0.18wt % of acetic acid so that a 0.4% chitosan solution could be prepared.Then, 7.5g of the 0.4 chitosan solution was added to 100 g of theresultant Bacillus natto culture in Example 1, and 2.5 g of pearlite wasadded thereto, and the mixture was stirred at room temperature for 10minutes. The mixture was filtrated under reduced pressure to give afiltrate. The activity of nattokinase and the amount of vitamin K2 inthe filtrate were determined as in Example 1. The results are shown inTable 1.

TABLE 1 Vitamin K2 *1 Nattokinase *2 Before After Removal Before AfterRetention No. Treatment filtration filtration (%) filtration filtration(%) Ex. 1 Disodium 5.7 below 100 480 475 99 hydrogenphosphate detectioncalcium chloride limit Ex. 2 Poly aluminum chloride 5.7 below 100 480478 99 detection limit Ex. 3 Sodium polyacrylate 5.7 0.1 98 480 464 97cationic coagulant of plant polysaccharides Ex. 4 Hexamethylenediamine/5.7 below 100 480 470 98 epichlorohydrin detection polycondensate limitEx. 5 Dimethylamine/ 5.7 below 100 480 470 98 epichlorohydrin detectionpolycondensate limit Comp. Chitosan 5.7 below 100 480 475 00 Ex. 1detection limit *1 μ/g culture *2 FU/ml culture

As evident from Table 1, almost all vitamin K2 was removed, whilenattokinase was retained in the Bacillus natto culture, with anytreatments in Examples 1 to 5, as in the case of conventionally knownchitosan.

According to the method of the present invention-, vitamin--K2 can beremoved from a Bacillus natto culture in a lower cost and a simplermanner than using chitosan. The thrombolytic activity of nattokinasewould function more effectively by reducing the amount of vitamin K2that should act on the blood coagulation system. Therefore, the methodof the present invention is useful in producing a health food having anexcellent efficacy.

Since the food product of the present invention contains little vitaminK2, it is possible to prevent the overintake of vitamin K2 and thethrombogenesis in healthy people. For the patients who are restricted tointake of vitamin K2, the food product of the present invention can besafely ingested to intake nattokinase.

Example 6

A Bacillus natto culture was prepared in the same manner as Example 1.To 20 kg of the Bacillus natto culture, 880 g of disodium hydrogenphosphate was added and mixed. The mixture was stirred extensively, andthen 660 g of a 35 wt % calcium chloride aqueous solution was added andmixed. Then, 540 g of pearlite were added thereto and mixed. Theresultant mixture was stirred at room temperature for 30 minutes. The pHof the mixture was adjusted to 7.2, and the mixture was filtrated underreduced pressure to give a filtration residue. The amount of vitamin K2in the filtration residue was determined according to the procedure Bdescribed above, and found to be 308.8 μg wet weight of the filtrationresidue.

To 1 kg of the filtration residue, 5 kg of n-hexane was added andstirred at 40° C. for 24 hours in order to extract vitamin K2. Then-hexane fraction (1^(st) n-hexane extract) was collected by filtration.To the filtrate residue, 4 kg of n-hexane was further added and stirredat 40° C. for 3 hours, and the n-hexane fraction (2^(nd) n-hexaneextract) was collected. The 1^(st) and 2^(nd) n-hexane extracts werecollected, evaporated to remove the n-hexane, and distilled at 50° C.,100 HPa to obtain a crude oil. The resultant oil fraction was heated at150° C. for 2 hours for deodorization. Thus, 95 g of an oily productthat contains vitamin K2 at 2766 μg/g was obtained. To the thus-obtainedoily product that contains vitamin K2, the following components wereadded to make the composition stated below, and mixed, and the mixturewas then freeze-dried to obtain a powdered product:

Oily product that contains vitamin K2 80.84 g Gum arabic 7.19 g Sodiumarginate 6.45 g Citric acid 0.81 g Sodium bicarbonate 0.81 g Ascorbylpalmitate 0.40 g Calcium chloride 0.29 g Tocopherol 0.04 g

According to the method of the present invention, vitamin K2 can becollected from a Bacillus natto culture at a lower cost and a simplermanner than using chitosan.

The invention may be embodied in other forms without departing from thespirit or essential characteristics thereof. The embodiments disclosedin this application are to be considered in all respects as illustrativeand not limiting. The scope of the invention is indicated by theappended claims rather than by the foregoing description, and allchanges which come within the meaning and range of equivalency of theclaims are intended to be embraced therein.

The invention claimed is:
 1. A method for producing vitamin K2,comprising: culturing Bacillus natto in a liquid medium to obtain aBacillus natto culture or culture supernatant thereof, wherein theculture or culture supernatant contains vitamin K2; treating theBacillus natto culture or culture supernatant with an inorganiccoagulant in combination with a phosphorus-containing coagulant, whereinthe inorganic coagulant is calcium chloride and thephosphorus-containing coagulant is at least one selected from the groupconsisting of disodium hydrogenphosphate, sodium dihydrogenphosphate,potassium dihydrogenphosphate and tripotassium phosphate, so as to forma coagulant to which vitamin K2 from the Bacillus natto culture orculture supernatant is absorbed or aggregated; separating the coagulantto which vitamin K2 is absorbed or aggregated from the treated cultureor culture supernatant; treating the coagulant to which vitamin K2 isabsorbed or aggregated with an organic solvent for extraction;collecting an organic solvent extract; removing the organic solvent fromthe organic solvent extract; and obtaining an oily product that containsvitamin K2 from the extract from which the organic solvent has beenremoved.
 2. The method of claim 1, wherein the separation is performedby filtration so as to obtain the coagulant to which vitamin K2 isabsorbed or aggregated as a filtration residue.